The Latest RBD 3000 ApplicationsThe Latest RBD 3000 Applications

Advanced Analytical User’s MeetingNovember 30, 2006

Chicago, IL

OverviewOverview

• Contamination Level Test – For materials that contain low-level bio-burden

– Determines that material meets microbial specification

– Utilizes the Most Probable Number (MPN) protocol

– Useful for complex matrices

• Antimicrobial Effectiveness Test– Rapidly screens candidate preservative systems

– Tracks an increase in dead cell count

Contamination Level Test – Contamination Level Test –

Feasible in a Variety of Complex MatricesFeasible in a Variety of Complex Matrices

Company A – In-process pharmaceutical product

Company B – Various nutraceutical finished products– Juice, Body Butter, Fiber

Company C – Liquid nutraceutical finished product

Company D – Various alcoholic beverages

CLT – Protocol for <100cfu/g specificationCLT – Protocol for <100cfu/g specification

CLT – Results Company ACLT – Results Company AIn-process Product: Specification <1000cfu/g

Sample DilutionRBD 3000 Result

(# pos./# neg.)

Plate Result

(# pos./# neg.)Microbial level determined by

RBD 3000 (cfu/g)

1:10 3/0 3/0 >1

1:100 3/0 3/0 >10

1:1,000 3/0 3/0 >100

1:10,000 1/2 1/2 <1,000

Sample DilutionRBD 3000 Result

(# pos./# neg.)

Plate Result

(# pos./# neg.)Microbial level determined by

RBD 3000 (cfu/g)

1:10 3/0 3/0 >1

1:100 3/0 3/0 >10

1:1,000 3/0 3/0 >100

1:10,000 3/0 3/0 >1,000

Spiked Below Specification

Spiked Above Specification

Product contains >100cfu/g and <1000cfu/g; product is within specificationActual spike was 559 cfu Ps. aeruginosa/g product

Product contains >1000cfu/g; product fails Actual spike was 3920 cfu Ps. aeruginosa/g product

CLT – Results Company BCLT – Results Company B

Product contains >100cfu/g; product failsActual spike was 110cfu C. albicans/mL juice

Taken from poster presentation at ASM 2006 A Rapid Most Probable Number Test for Yeast in Nutraceutical Products, K.A. Molitor and A.M. Steger, Advanced Analytical Technologies, Inc., Ames, IA 50010 and D. Wright and M. Roblin, Morinda, Inc., Orem, UT 84604

Sample DilutionRBD 3000 Result

(# pos./# neg.)

Growth on YM PetrifilmTM

(# pos./# neg.)

Microbial level determined by RBD 3000 (cfu/g)

1:100 2/1 2/1 >10

1:1,000 0/3 0/3 <100

1:10,000 0/3 0/3 <1000

Sample DilutionRBD 3000 Result

(# pos./# neg.)

Growth on YM PetrifilmTM

(# pos./# neg.)

Microbial level determined by RBD 3000 (cfu/g)

1:100 3/0 3/0 >10

1:1,000 2/1 2/1 >100

1:10,000 1/2 1/2 <1000

Body Butter & Juice Products: Specification <100cfu/g or mL

Juice: Spiked Above Specification

Body Butter: Spiked Below Specification

Product contains >10cfu/g and <100cfu/g; product is within specificationActual spike was 35 cfu C. albicans/g body butter

Contamination Level Test – BenefitsContamination Level Test – Benefits

• 18-24hr time-to-result

• Detects lower levels of potential microbial contamination

than the traditional plate count method in complex/opaque

matrices

• Results are confirmed by the “Most Probable Total Count

by Multiple-Tube Method” in USP Chapter <61>, Microbial

Limits Test

Antimicrobial Effectiveness TestAntimicrobial Effectiveness Test

Antimicrobial Effectiveness TestAntimicrobial Effectiveness Test

• Used to screen candidate preservatives for liquid products

• Pharmaceutical and Cosmetic Industries routinely perform

– New & reformulated products

• Problems with the current method:– Compendial method takes 35 days

– Requires significant labor & materials

– Not all types &/or formulations of antimicrobials are screened due

to time & labor constraints

– Delays development of suitable preservative systems

– Delays product time to market

Antimicrobial Effectiveness Test – Antimicrobial Effectiveness Test – SolutionSolution

• Use the RBD 3000 to rapidly screen candidate

preservative systems

• Benefits:

– Shorter time to result

– Significant labor and materials savings

– Evaluate more types/formulations of antimicrobials

– Develop better preservative systems

– Speed product time to market

Antimicrobial Effectiveness Test – Antimicrobial Effectiveness Test – ProtocolProtocol

• Challenge 0.01% (w/v) Benzoic Acid with 105 – 106 cfu/mL

• Perform challenge studies with Ps. aeruginosa, S. aureus,

and C. albicans

• Use RBD 3000 to determine titer of inoculum

• Sample at 0, 2hr, 4hr, 6hr, 8hr & 24hr post-inoculation– Dilute samples 1:10 in phosphate buffer for RBD 3000 Biomass &

Dead Cell counts using the Biomass and Dead Cell Test Kits

– Dilute samples in phosphate buffer + 4% Tween 20 for TSA plate

count comparison

0.01% Benzoic Acid Challenged with0.01% Benzoic Acid Challenged withPs. aeruginosaPs. aeruginosa – Test 1 – Test 1

5.76 5.83 5.83 5.84 5.83 5.81

4.14

4.684.93

5.14 5.22

5.735.785.64

5.345.12

5.29

3.98

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24

Time post-inoculation (hours)

RB

D 3

000

log

10 C

ou

nts

/mL

Ps .aeruginosa-Biomass Count Ps. aeruginosa-Dead Cell Count Ps. aeruginosa-Plate Count

0.01% Benzoic Acid Challenged with0.01% Benzoic Acid Challenged with Ps. aeruginosaPs. aeruginosa – Test 2 – Test 2

5.72 5.76 5.75 5.75 5.77 5.77

4.46

4.865.10

5.27 5.36

5.715.59

5.465.25

5.10 5.18

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24

Time post-inoculation (hours)

RB

D 3

00

0 lo

g 10 C

ou

nts

/mL

Ps. aeruginosa-Biomass Count Ps. aeruginosa-Dead Cell Count Ps. aeruginosa-Plate Count

2.4

0.01% Benzoic Acid Challenged 0.01% Benzoic Acid Challenged withwith S. aureus S. aureus

5.59 5.61 5.62 5.61 5.60 5.58

5.145.37 5.47 5.52 5.53 5.59

5.965.81

5.50

5.174.94

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24Time Post-Inoculation (hours)

RB

D 3

000

log

10 C

ou

nts

/mL

S. aureus-Biomass Count S. aureus-Dead Cell Count S. aureus-Plate Count

1.78

0.01% Benzoic Acid Challenged 0.01% Benzoic Acid Challenged withwith C. albicans C. albicans

5.115.24 5.31 5.31 5.25

5.37

3.93 4.03 4.00 3.983.79

4.36

5.55 5.45 5.45 5.44 5.53 5.56

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24

Time Post-Inoculation (hours)

RB

D 3

00

0 lo

g 10

Co

un

ts/m

L

C. albicans-Biomass Count C. albicans-Dead Cell Count C. albicans-Plate Count

Antimicrobial Effectiveness Test – Antimicrobial Effectiveness Test – Action ItemsAction Items

• Test other preservative systems

• Determine utility in various liquid product formulations

• Demonstrate feasibility with A. niger spores

SummarySummary

• Contamination Level Test – Provides a rapid & sensitive detection method

for determining if materials meet particular

microbial specifications

• Antimicrobial Effectiveness Test– Provides a rapid method for screening candidate

preservative systems